Demonstration of putative subunit(s) of a novel insulin receptor-associated 66 kDa GTP-binding protein, Gir

A 66 kDa GTP-binding protein, Gir, and insulin receptor (IR) were copurified from human placental membrane by DEAE-Sephacel and Wheat Germ Agglutinin (WGA)-Sepharose affinity chromatography. The WGA-fraction containing IR and Gir (IR-Gir-fraction) was phosphorylated (95 kDa IR-beta and 66 kDa Gir) by IR-tyrosine kinase using [32P]ATP or photolabeled with [32P]8-azido-GTP (mainly 66 kDa), and was cross-linked with a bifunctional reagent, bis-[sulfosuccinimidyl] suberate (BS3). The Gir cross-linked with putative subunit(s) to form a 110 kDa complex. Phosphorylation as well as 8-azido-GTP binding to Gir was not affected by cross-linking indicating that like other G-Proteins, Gir may also have subunits and that cross-linking of Gir with its putative subunits(s) does not block GTP-binding and phosphorylation sites.